Journal of Traditional and Complementary Medicine xxx (2018) 1e6

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Journal of Traditional and Complementary Medicine

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Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton

flower extract on isolated rat ileum contractions and the mechanisms
of action
Acharaporn Duangjai a, b, *, Bey-Hing Goh b, c, Learn-Han Lee b, c, Surasak Saokaew b, c, d
a
Division of Physiology, School of Medical Sciences, University of Phayao, Phayao, Thailand
b
Center of Health Outcomes Research and Therapeutic Safety (Cohorts), School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand
c
School of Pharmacy, Monash University Malaysia, Selangor Darul Ehsan, Malaysia
d
Center of Pharmaceutical Outcomes Research (CPOR), Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Azadirachta indica A. Juss var. siamensis Valeton or commonly known as Siamese neem is one of the most
Received 13 September 2016 well-known plant in traditional Ayurvedic medicine. The aim of the present study was to investigate the
Received in revised form relaxant effects of A. indica on isolated rat ileum contractions and its potential underlying mechanisms
11 December 2017
involved. The isometric contractions of ileum segments were investigated in organ baths for spontaneous
Accepted 2 January 2018
Available online xxx
activity and response to aqueous extract of Siamese neem flower (SNF). The spasmolytic action of the
extract was also assessed on contraction induced by acetylcholine and high potassium. Our findings
indicate that cumulative concentrations of SNF aqueous extract induced relaxant effect on spontaneous
Keywords:
Siamese neem
rat ileum contractions. The extract has also suppressed the cumulative concentration response curve for
Ileum acetylcholine and pottasium ions-induced contraction. The presence and absence of propranol (antag-
L-name onist of b-adrenergic receptor) and L-Name (antagonist of nitric oxide synthase) in SNF aqeous extract co-
Propanolol treatment demonstrated no significant different in term of contraction activity when compared to SNF
CaCl2 extract treatment alone. The treatment of SNF extract caused a significant inhibition in tissue contraction
stimulated by accumulation of calcium ions. Our results showed the relaxant effect of SNF aqueous
extract on the isolated rat ileum. In short, the SNF aqueous extract exhibited an inhibitory effect on the
spontaneous ileum contactions particularly on the contraction stimulated by acetylcholine and high
potassium. The observed effect might acted through the modulation of calcium channels. This findings
provide a pharmacological basis for the traditional use of SNF for the treatment of gastrointestinal
spasms.
© 2018 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier
Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/4.0/).

1. Introduction GI functional and motility disorders.2 Although the etiology of

Gastrointestinal (GI) diseases and symptoms are known to cause
an effect on quality of life, work and activity impairments, and
health care costs.1 A disturbance in coordination between the
motor, sensory or secretory function of GI tract are known to cause
* Corresponding author. Division of Physiology, School of Medical Sciences,
University of Phayao, Phayao 56000, Thailand.
E-mail addresses: achara.phso@gmail.com (A. Duangjai), goh.bey.hing@monash.
edu (B.-H. Goh), lee.learn.han@monash.edu (L.-H. Lee), saokaew@gmail.com,
surasak.sa@up.ac.th (S. Saokaew).
Peer review under responsibility of The Center for Food and Biomolecules,
National Taiwan University.
some cases of GI functional and motility disorders are still remain
unclear, the common observed symptoms are including nausea,
vomiting, bloating, abdominal discomfort or pain, constipation or
diarrhea.2 The onset of GI motility disorders has been attributed to
several causes, which including the consequence complications of
other systemic illnesses, such as diabetes.2,3
Generally, the pharmacological interventions for GI motility
disorders are involved in altering the regulation of GI smooth
muscle, enteric nervous system (ENS), autonomic ganglia, and
central nervous system. Radulovic M et al. have suggested that
release of neurotransmitters and several ion channels in GI tissues
including ATP-sensitive potassium (KATP) channel, calcium ions

https://doi.org/10.1016/j.jtcme.2018.01.003
2225-4110/© 2018 Center for Food and Biomolecules, National Taiwan University. Production and hosting by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Duangjai A, et al., Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton flower extract on isolated
rat ileum contractions and the mechanisms of action, Journal of Traditional and Complementary Medicine (2018), https://doi.org/10.1016/
j.jtcme.2018.01.003
2 A. Duangjai et al. / Journal of Traditional and Complementary Medicine xxx (2018) 1e6
(Ca2þ)-activated potassium ions (Kþ) channels, voltage-sensitive
Ca2þ channels and chloride ion channels play an important role
in regulating GI motility.4 In fact, those mechanisms have served as
the fundamental knowledge in new discoveries and development
of new therapeutic agents. Cholinergic agonists or laxative agents
are widely used for gastrointestinal motility disorders manage-
ment.4 However, these drugs have an undesirable side effect.
Complementary and alternative medicines (CAM) have been asso-
ciated with symptom management and quality of life in a common
gastrointestinal disorder.5 Therefore, the attention has been shifted
to bio-prospecting the natural products to overcome or control GI
motility disorders.
For centuries, herbs have been used in traditional medicine to
treat GI disorders. With the recent advancement of technologies or
scientific techniques, it enables the essences of this traditional
knowledge to be explored further. These investigations have
opened up more avenues which either allows a more systematic
usage of traditional medicine or alternatively to be developed to
become a standard drug. For instance, Siamese neem tree (Azadir-
achta indica A. Juss. var. siamensis Valeton) which is belong to
Meliaceae family has been used in Ayurvedic medicine for more
than four thousand years.6 All parts of the neem tree (leaves,
flowers, seeds, fruits, roots and bark) have been used for the
treatment of inflammation, infections, fever, skin diseases and
dental disorders.7 The robustness and well-studied biological ac-
tivities has thus contributed to its commercial exploitation for
treatment of various diseases. In particular, it is traditionally sug-
gested and used to treat diarrhea and peptic ulcer.8e11 However, to
the best of our knowledge the role of Siamese neem flower (SNF) on
intestinal smooth muscle motility is remaining unclear. Therefore,
the current study aims to study the effect of SNF extract on isolated
rat ileum contractions induced by high potassium and acetylcho-
line, and investigated the possible mechanisms involved. The
findings might help to explain the extensively usage of SNF for the
treatment of gastrointestinal diseases.
2. Materials and methods
2.1. Chemicals
Atropine sulphate, propranolol, acetylcholine chloride and L-
NAME were purchased from Sigma (USA), KCl, CaCl2 and other re-
agents were obtained from Merck Company (Germany). The extract
and chemicals were dissolved in distilled water for experimenta-
tion. The Krebs' solution used in these experiments had the
following composition: 122 mM NaCl, 5 mM KCl, 10 mM HEPES,
0.5 mM KH2PO4, 0.5 mM NaH2PO4, 1 mM MgCl2, 1.8 mM CaCl,2 and
11 mM glucose; pH 7.3.
2.2. Plant materials
Siamese neem flowers (SNF) were collected from Tumbon
BanMaetumboonyong Mueang Phayao, Thailand. The plant was
identified by a botanist and a voucher specimen (No. 003805) was
deposited at the herbarium of the Faculty of Biology, Naresuan
University, Phitsanulok, Thailand.
2.3. Preparation of SNF aqueous extract
Siamese neem flowers were washed, cut in smaller pieces, and
blended in an electric blender in distilled water (plants 100 g: water
300 mL). They were filtered to obtain a crude solution and subse-
quently freeze-dried by freeze dryer (Scam VacCoolSafe 110e4 Pro)
until powder was obtained. The lyophilized sample powder or
extract was then stored at 20  C. Prior to experimentation, the
Please cite this article in press as: Duangjai A, et al., Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton flower extract on isolated
rat ileum contractions and the mechanisms of action, Journal of Traditional and Complementary Medicine (2018), https://doi.org/10.1016/
j.jtcme.2018.01.003
powder extract was prepared by dissolving it in appropriate
amount of distilled water.
2.4. Animals
Male Wistar rats (200e250 g) were obtained from the National
Laboratory Animal Centre, Mahidol University, Salaya, Nakhorn-
pathom, Thailand. Experiments were approved by the Animal
Ethics Committee of University of Phayao, Phayao, Thailand. Ani-
mals were maintained at Laboratory Animal Research Center, Uni-
versity of Phayao under 12-h dark/12-h light condition and free
access to water and standard rodent diet.
2.5. Tissue preparation and experimental procedure
After animals were fasted overnight, they were deeply anes-
thetized by 50 mg/kg BW zolazepam/tiletamine (zoletil) and 3 mg/
kg BW xylazine. Two pieces of ileum were isolated from 2 cm above
the ileocaecal junction. The intraluminal content was flushed out
with cooled Krebs' solution and cleaned off connective tissue sur-
rounding. The tissue was mounted in an organ bath (30 mL) (the
lower region was tightly tied to the bottom inside the bath while
the upper region was connected to an isotonic force transducer)
containing Krebs'solution and maintained at 37  C, pH7.4, and a
continuous supply of oxygen from air bubbles. The ileum was
equilibrated for 1 h under 1 g resting and the solution was replaced
every 15 min. Ileum contraction were measured using force trans-
ducer connected to an iWorx214 A/D converter (LabScribe2; In-
struments, Thailand) and the data was recorded.
2.6. Relaxation effects of siamese neem flower
To examine the relaxation effect of SNF on ileum contraction,
SNF extract (0.01e10 mg/mL) was cumulatively added into the or-
gan bath once the contraction plateau was reached by the KCl
(80 mM) induction. The contractions induced by the extract was
recorded and normalized to KCl induced activity which was
considered as the maximum contraction (100% contraction). In
order to investigated the mechanism of action involved in relaxant
effect demonstrated by SNF, experimentations with ileum pre-
incubation in 100 mM L-Name (antagonist of nitric oxide synthase)
for 20 min or 1 mM propanolol (antagonist of b-adrenergic receptor)
for 30 min prior to KCl 80 mM exposure were then conducted.
Following the preincubation period as stated above, the extract
(5 mg/mL) was then added into the organ bath when KCl induced
contraction reached a constant activity. On the other hand, the
addition experiments were also conducted in order to examine the
acetylcholine chloride (105 M, agonist of acetylcholine receptor)
induced ileum contraction activity in either the presence or
absence of extract (5 mg/mL).
2.7. Effect of siamese neem flower on extracellular Ca2þ influx
In order, to determine the role of SNF on extracellular calcium,
Ileum was initially equlibrated in Ca2þ free Krebs' solution
(0.01 mM EGTA, 122 mM NaCl, 5 mM KCl, 10 mM HEPES, 0.5 mM
KH2PO4, 0.5 mM NaH2PO4, 1 mM MgCl2, and 11 mM glucose; pH
7.3) for 30 min. Following the incubation, SNF extract was added
into the solution. Subsequently, a cumulatively concentration of
CaCl2 (1e40 mM) was then added into the organ bath to stimulate
the contraction. The contraction activity was observed and recor-
ded for 10 min following the addition. The activity was determined
by comparing the extract presence group to the control group or
known as extract absence group.
 A. Duangjai et al. / Journal of Traditional and Complementary Medicine xxx (2018) 1e6 3

2.8. Statistical analysis
Data was expressed as Mean ± SEM (n ¼ 6e8 for each set of
experiments). For data with normal distribution, the Analysis of
variance (ANOVA) was used to test of significant difference be-
tween the groups if there were more than 2 groups. Bonferroni
multiple-comparison test was also used for post-hoc analysis. If
data were skewness, Kruskal-Wallis test were used to test the dif-
ference and multiple-comparison among 3 groups. . P-values less
than 0.05 were considered statistically significant.
(5 mg/mL) induced relaxation in the presence and absence of L-
Name (Fig. 2A). There were no statistically significant differences of
ileum relaxtion between the presence and absence of L-Name. The
relaxation effects of the extract responsed to 80% and 71%of ileum
contraction induced by the presence and absence of L-Name,
respectively. Similarly, preincubating of propranolol, the contrac-
tion was attenuated significantly in the presence of the extract
(Fig. 2B). Moreover, pretreatment of the SNF extract (5 mg/mL) and
atropine (100 nM) showed a significantly abolished the
acetylcholine-effect (1  105 M) as shown in Fig. 3.

3. Results 3.2. Effect of siamese neem flower on extracellular Ca2þ influx

3.1. Effects of siamese neem flower extracts on ileum relaxations
In order to study whether SNF extract reduced ileum contrac-
tions, 80 mM KCl was used for induction of the contraction. The
cumulative concentrations of SNF extract (0.01e10 mg/mL) was
added after KCl induced contraction reached to the plateau states.
The extracts produced ileum relaxations in a dose dependent
manner induced by KCl (Fig. 1). The extract at 5 and 10 mg/mL
responsed to 71% and 68% of the contraction, respectively. How-
ever, there were no statistically significant difference of ileum
contractions between 5 and 10 mg/mL of the extract. Therefore,
5 mg/mL of the extract was used in the subsequent study to define
its potential mechanisms. To investigated the mechanism involved
of the SNF extract on ileum relaxation, ileum was pretreated with
100 mM L-Name (antagonist of nitric oxide synthase) or 1 mM
propanolol (antagonist of b-adrenergic receptor). The extract
To investigated whether SNF extract produced ileum relaxation
by interfering Ca2þ influx, the cumulative concentrations of CaCl2
(1e40 mM) were added in the bath containing Ca2þ free solution
containing high Kþ in the presence or absence SNF extract (5 mg/
mL). The effect of Ca2þ induced ileum contraction in the absence
and presence SNF extract on ileum contraction are shown in Fig. 4.
Ca2þ induced a progressively increased the contraction in the
depolarized tissue through KCL (80 mM). The contraction induced
by CaCl2 was significantly decreased in the presence of SNF extract
approximately 29%, 43%, and 45% at CaCl2 concentrations 10, 20,
and 40 mM, respectively (Fig. 4B).
4. Discussion
The regulation of gastrointestinal motility and smooth muscle
activity depends on hormones neurotransmitters, and numerous

Fig. 1. Effects of cumulative concentrations of SNF extract on rat's ileum contractions induced by KCl. 80 mM KCl were added to an organ bath to induce constant contractions. The
extract (0.01e10 mg/mL) was then added cumulatively. (A) Representative trace of SNF extract and (B) summary of inhibitory effect of SNF extract on ileum contractions induced by
KCl. Values are means ± SEM for 8 experiments. *Significantly different from values for control (P < .001).

Please cite this article in press as: Duangjai A, et al., Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton flower extract on isolated
rat ileum contractions and the mechanisms of action, Journal of Traditional and Complementary Medicine (2018), https://doi.org/10.1016/
j.jtcme.2018.01.003
4 A. Duangjai et al. / Journal of Traditional and Complementary Medicine xxx (2018) 1e6
Fig. 2. The contractile effects of KCl on ileum and the inhibitory effect of SNF extract
and (A) L-Name or (B) propanolol on KCl-induced contraction. Ileum was preincubated
with 100 mM L-Name (antagonist of nitric oxide synthase) for 20 min or 1 mM prop-
anolol (antagonist of b-adrenergic receptor) for 30 min prior to KCl 80 mM. Data are
expressed as means ± SEM for 8 experiments. Panel (A), * significantly different among
3 groups compared with High Kþ (P < .001). Panel (B), significantly different between
propanolol and/or SNF extract compared with High Kþ (#P < .001 and @P ¼ .021,
respectively).
Fig. 3. Effect of SNF extract and atropine on acetylcholine-induced contraction. Ileum
was preincubated with the extract (5 mg/mL) or atropine (100 nM) for 20 min prior to
acetylcholine chloride (105 M). Values are means ± SEM for 6 experiments. SNF
extract or atropine significantly different compared with acetylcholine (*P ¼ .002 and
**P ¼ .001, respectively).
mediators including acetylcholine, noradrenaline and non-
adrenergic non-cholinergic (NANC), which can be excitatory or
inhibitory to gastrointestinal smooth muscle.12,13 The purpose of
Please cite this article in press as: Duangjai A, et al., Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton flower extract on isolated
rat ileum contractions and the mechanisms of action, Journal of Traditional and Complementary Medicine (2018), https://doi.org/10.1016/
j.jtcme.2018.01.003
this study was to investigate the relaxant effects of siamese neem
flower (A indica A. Juss var. siamensis Valeton) on rat ileal smooth
muscle contractions.
KCl and acetylcholine (ACh) are known to induce smooth muscle
contraction. The high level of Kþ induces cell depolarization and
consequently activates of L-type voltage-dependent calcium
channels. The activation of calcium channels will cause the increase
of Ca2þ influx into smooth muscle cell which eventually induced
contraction.14 Meanwhile, Ach has known to play roles in the
excitatory effect of parasympathetic nerves on intestinal smooth
muscle. It binds to muscarinic receptors on the smooth muscle cell
membrane15 and thereby increases the activity of phospholipase C
which generates inositol triphosphate (IP3). The release of IP3 from
the plasma membrane allowed it to stimulate the release of intra-
cellular Ca2þ stores and subsequently induce contraction of smooth
muscle cells.
However, under the influence of the competitive antagonist of
muscarinic receptors such as atropine will lead to the response in
inhibition of contraction. Therefore, substances that inhibit the Kþ
induced contraction may exert its effect through the blockage of the
Ca2þ channel or preventing the entry of Ca2þ into smooth muscle
cell. In our experiments, the presence of SNF extract significantly
reduced the rat ileum contractions induced by KCl and ACh.
Furthermore, SNF extract has also showed the atropine-like action
by abolishing the stimulatory effect of acetylcholine. It could be
possibly due to the fact that, SNF extract may possess an action as
Ca2þ channel blocker or antagonist agent to ACh.
In order to further examine the relaxant effect of SNF extract,
CaCl2 were then utilized in following study. Interestingly, the
presence of SNF extract was observed to cause a significant
decrease in ileum smooth muscle contraction induced by CaCl2. The
result of the study implies the relaxant effect possess by SNF extract
might acted through the blockage of calcium channel. The findings
are found in agreement with many other studies, as plants such as
Celastrus paniculatus16, Curcuma aeruginosa Roxb.17, and Rosmarinus
officinalis L18 are known to exhibit calcium channel blocker-like
activity.
Since nitric oxide (NO) and b-adrenoceptors are also known to
affect gastrointestinal motility, the possible involvement of those
receptors in observed SNF relaxant effect was then examined
further as well. NO is synthesized from the L-arginine by nitric
oxide synthase and it plays a major roles as an inhibitory NANC
mediator in the gastrointestinal tract.12 It induces smooth muscle
relaxation and hyperpolarization by activating the soluble guany-
late cyclase and leading to the formation of cyclic guanosine
monophosphate (cGMP). The increase in cGMP was then leads to
the activation of cGMP dependent protein kinase G, which not only
dephosphorylated the myosin light-chain but also activates po-
tassium channels, and on the same time reducing the intracellular
calcium concentrations.12,19 Therefore, the further study was con-
ducted in order to understand whether the relaxant effect
demonstrated by SNF extract might have acted through the mod-
ulation of nitric oxide production. However, the results showed that
ileum relaxation induced by SNF extract was insensitive towards
the stimulation of L-Name, a nitric oxide synthesis inhibitor. In
addition, the simultaneous addition of L-Name and SNF extract
resulted in a similar trend of response which was observed in SNF
extract treatment group. It can be suggested that the relaxation
response is not mediated through NO pathway. Meanwhile,
another aspect of the study was focusing on the investigation on
the possible mediation of b-adrenergic receptor in SNF extract
stimulated relaxant effect. However, the experiment found there is
no real correlation between the b-adrenergic receptor and relaxant
effect showed by SNF extract. It was proven true as the results have
shown no statically difference between the reduction of ileum
 A. Duangjai et al. / Journal of Traditional and Complementary Medicine xxx (2018) 1e6 5

Fig. 4. Effects on ileum contraction induced by CaCl2 in the absence (Control) and presence of SNF extract. (A) Representative traces of effects of absence (i) and presence of SNF
extract (ii) and (B) summary of inhibitory effect of the extract on iluem contraction induced by cumulative Ca2þ in Ca2þ free solution containing 80 mM Kþ. Ileum was equlibrated in
Ca2þ free Krebs' solution prior to incubate with the SNF extract (5 mg/mL), subsequently treated with cumulative doses of CaCl2 (1e40 mM) into the organ bath to stimulate the
contraction. Values are means ± SEM for 8 experiments. *Significantly different from values for control (P < .001).

contraction induced by SNF extract either in the presence or extract.

absence of propranolol, a b-adrenergic receptor antagonist. The
results rule out the roles of b-adrenergic pathway in the relaxant
effect of SNF extract.
Studies have showed the phytochemical constituents of many
plants are able to attenuate the smooth muscle motility.20e22 Neem
tree (Azadirachta indica A. Juss. var. siamensis Valeton) contains
diterpenoids, triterpenoids, limonoids, proteins, polysaccharides,
sulphurous compounds, polyphenolics, dihydrochalone, coumarin
and tannins and aliphatic compounds7 alkaloids, flavonoids, ste-
roids, gum, carbohydrates, saponins, and tannin.23 b-sitosterol,
lupeol, rutin, ellagic acid, ferulic acid and quercetin,6 azadirachtin,
nimbin, gallic acid, salanin, nimbolin, azadiradione, azadirone,
mimbinin.7 It have been reported that phenolic compounds such as
diterpene polyester and quercetin were involved in inducing ileal
smooth muscle relaxation.20e22 On the other hand, flavonoids such
as apigenin, genistein, quercetin, rutin, catechin, naringenin have
known to exhibit relaxant effect on isolated gastric stomach.24
Furthermore, the flavonoid known as galetin 3,6-dimethyl ether
has shown to decrease guinea pig ileum contraction through Kþ
channel activation and decrease in cytosolic calcium concentra-
tion.25 Therefore, the relaxant effect demonstrated by SNF might
possibly due to the presence of these bioactive compounds in the
However, further studies are needed to confirm bioavailability
of its active compounds and in vivo studies will also be required to
figure out the optimal dose for effective therapy.
5. Conclusions
The present study provides evidence that an aqueous extract of
siamese neem flower possesses a relaxant effect in isolated rat
ileum. The observed effect might have mediated through calcium
channels. The findings of current study provide a pharmacological
basis for the traditional use of SNF for the treatment of gastroin-
testinal spasms.
Conflicts of interest
The authors declare that they have no conflicts of interests.
Acknowledgements
The authors are very grateful Natakorn Kamkaew for support
the instrument and wish to acknowledge School of Medical Science,
University of Phayao for available to do research.

Please cite this article in press as: Duangjai A, et al., Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton flower extract on isolated
rat ileum contractions and the mechanisms of action, Journal of Traditional and Complementary Medicine (2018), https://doi.org/10.1016/
j.jtcme.2018.01.003
6 A. Duangjai et al. / Journal of Traditional and Complementary Medicine xxx (2018) 1e6

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Please cite this article in press as: Duangjai A, et al., Relaxant effects of Azadirachta indica A. Juss var. siamensis Valeton flower extract on isolated
rat ileum contractions and the mechanisms of action, Journal of Traditional and Complementary Medicine (2018), https://doi.org/10.1016/
j.jtcme.2018.01.003